determination of sucrose in cane juice and [PDF]

The sucrose was extracted from beet with di-ethylamine and was crystallised from this solvent by addition of ethanol. Th

3 downloads 3 Views 370KB Size

Recommend Stories


SUCROSE
You often feel tired, not because you've done too much, but because you've done too little of what sparks

sucrose
Sorrow prepares you for joy. It violently sweeps everything out of your house, so that new joy can find

Melatonin in Apples and Juice
Suffering is a gift. In it is hidden mercy. Rumi

View CEO Juice PDF
Open your mouth only if what you are going to say is more beautiful than the silience. BUDDHA

Determination of proteins in the presence of Phenol, Sucrose, Mannitol, Glucose, Fructose, and Tris
Seek knowledge from cradle to the grave. Prophet Muhammad (Peace be upon him)

Determination of Carbendazim and Benomyl Residues in Oranges and Orange Juice by
Don’t grieve. Anything you lose comes round in another form. Rumi

The Spectral Photometric Determination of Sucrose in Sugar Beets and Sugar Beet Products Via
The happiest people don't have the best of everything, they just make the best of everything. Anony

The Determination of Fruit Juice Authenticity Using High
Be grateful for whoever comes, because each has been sent as a guide from beyond. Rumi

Idea Transcript


Proceedings of The South Afvican Sugar Technologists' Association-June 1973

44

DETERMINATION OF SUCROSE IN CANE JUICE AND MOLASSES BY ISOTOPE DILUTION By J. BRUIJN and R. A. CARREYETT Sugar Milling Research Institute Abstract

Sucrose in first expressed juice and mixed juice was determined by isotope dilution and the results were compared with the conventional pol determination. i t was found that the sucrose content on the average was 0,25% higher using the I D method. Molasses samples, on the contrary, showed a sucrose content which was 0,71% lower than the value found by the Mackay method. The same molasses samples were also analysed by gas chromatography which gave a value 2,24% lower than the Mackay method. 1. Introduction Most conventional analytical methods for the determination of sucrose in factory products depend upon polarisation. These measurements are unreliable i n the presence of optically active non-sucrose constituent~.~-~ Although many modifications in the polarisation methods have been introduced in order to minimise interference, none is completely reliable, according to Hir~chrnuller.~ The latter author5 in 1959 introduced a method for sucrose determination in sugar beet based on isotope dilution and this is free from the above-mentioned types of interference. In isotope dilution methods, a measured quantity of labelled sucrose is added to the sample under investigation, after which sufficient pure sucrose is isolated from the sample to establish a radio-active count. From the ratio between the radio activity of the standard added and of the finally recovered sucrose, the original sucrose concentration in the sample can be calculated, taking into account the amount of labelled sucrose which was added to the sample. in the present investigation the isotope dilution method was applied to cane juice and cane molasses. The methods which have been used for sugar beet and beet sugar products were studied and modified for application to cane sugar products. The values for sucrose content in juices and molasses as obtained by the isotope dilution method were compared with those obtained by conventional methods. 2. Technique of radio active measurement In the case of a labelled sucrose molecule, either the C-atom or the H-atom can be labelled. The C-atom has the advantage that 14C has a long half life (5 570 years) and no corrections have to be made for radio-active decay during the determination. In addition to this the C-atom is built into the skeleton of the sucrose molecule, while the H-atom can wander through the molecule and be lost by dissociation during the determination.

The radiation of 14Cis very weak - 14C+ 14Nt P(0,154 MeV) - and special measuring techniques have to be used. Hirschmiiller and Hoerning5 used a proportional gas flow counter, operated with methane. In this type of counter, there is no mica window separating the sample from the Geiger-Muller (G.M.) tube, as the sample is placed inside the G.M. tube. In this way a reasonable counting efficiency is obtainable. Higher counting efficiencies can be obtained in a liquid scintillation counter. In this system, the radio-active material is dissolved or dispersed in a mixture of organic components which emit photons in the ultra violet region of the spectrum when it is energised by radio-active radiation. These photons are counted by a photo multiplier tube. With the earlier scintillation counters reasonable efficiency was only obtained at low temperature, which limited the application of this technique. Present instruments, however, are less sensitive to thermal excitation and counting at ambient temperature is standard practice. Nevertheless it was found during this investigation that background counts started to increase above 25°C. For this reason the temperature of the laboratory which housed the scintillation counter was kept below 25°C. 3. Methods for the isolation of sucrose from the sample As mentioned in the introduction, the I D method requires that a certain quantity of 100 % pure sucrose is isolated from the sample. Hirschmuller and Hoerning5 made use of the sucrose-copper complex for this isolation. The sucrose was extracted from beet with di-ethylamine and was crystallised from this solvent by addition of ethanol. The crude sucrose was dissolved in a copper sulphate solution and after one hour the copper-sucrose complex was separated from this mixture. The complex dissociates in hot water and the copper was removed by the addition of collidine, resulting in an insoluble copper-collidine complex. The filtrate of this solution was colourless and clear and pure sucrose was crystallised by the addition of ethanol. This method was modified in 1970 by Gelen.6 The beet pulp was extracted with a basic lead acetate solution and filtered. The filtrate was passed through a cation and anion exchanger, concentrated under vacuum and the sucrose crystallised by the addition of ethanol. This crude sucrose was then purified as described previously by forming a copper-sucrose complex after which the copper was removed by collidine. This modification was claimed to be less time-consuming and it gave 'results identical to the original method developed by Hirschmuller. Gelen also modified the counting technique by using a

Proceedings of The South Afvican Sugar Technologists' Association-

liquid scintillation counter, which was applied by McGagin and Eis in 1968' for counting 14C sucrose and is at present generally used for counting radioactive carbon and hydrogen. A different method of purification was proposed by Sibley and co- worker^.^ They purified the sucrose by preparing the barium saccharate and then decomposed the latter by bubbling carbon dioxide through the barium saccharide suspension. The suspension was subsequently filtered through sintered glass to remove the barium carbonate formed. The sucrose was further purified in the same way described for the other methods. Mauchg compared the method of Hirschmiiller and Hoerning with that of Sibley and co-workers for the determination of sucrose in beet. During the initial tests Mauch found that the method of Hirschmuller gave higher sucrose values in beet than the method of Sibley. Additional experiments showed that in Sibley's method the sucrose was not completely extracted from the beet pulp. After increasing the amount of water in the mixture used for the extraction of the beet pulp the two methods gave sucrose values which agreed within the errors to be expected. 4. Experimental In the earlier part of this investigation the standard sucrose was prepared as described by McGagin and Eis.' At a later stage a modification of Lew1° was followed. The 14C sucrose used was Calatomic* sucrose - U-C-14 50pc dissolved in 0,125 ml 25% ethanol. Using these vials procedure 1, described below (4.1.1) was followed. If after a number of analyses sufficient radio-active sucrose had been collected this was reprocessed into standard sucrose following procedure 2. 4.1.1 Procedure 1 Fifty grams of AR sucrose were dissolved in 12,5 ml distilled water. The mixture was heated to 85-90°C and maintained at that temperature. When all the sugar was dissolved the contents of two vials 14C sucrose (100 pc) were added and the mixture was stirred for 15 minutes after which it was filtered through a millipore filter (0,l PC). The filter was washed and the wash water was combined with the residues of previous determinations and reprocessed according to procedure 2. To the filtrate ethanol was added and the crystals so recovered were again recrystallised twice from ethanol. The final product was dried under vacuum at 6570°C. The number of counts per minute on 0,2 000 g were determined and if the value was not between 380 000 - 420 000 the radio-activity was adjusted by adding to the redissolved crystals either more non radio-active sucrose or 14C-sucrose as required. 4.1.2 Procedure 2 The residues of previous determinations, all having a radio activity of about 200 000 counts per minute *Los Angeles, Calif. 90054.

per 0,2 000 g, were added to 50 g of AR sucrose dissolved in 12,5 ml of distilled water. Sufficient 14Csucrose was added to raise the activity to the required level and the solution was processed as described under 4.1.1. 4.2.1 Isolation and pur$cation of sucrose from cane juice In the present investigation the method of Hirschmuller was tried first. It was, however, found difficult to obtain a completely copper-free solution after the precipitation of the copper-collidine complex. In addition, the method was rather elaborate. Subsequently the juice was first clarified by the addition of dry basic lead acetate. After filtration the lead was precipitated by hydrogen sulphide followed by a second filtration. The clear filtrate was further purified by passing it successively through a cation and anion exchange column. The deionised solution was concentrated under vacuum in a rotary evaporator to a syrup, absolute ethanol was added and the solution was cooled in a refrigerator. The crystallised sucrose was dissolved and recrystallised after which it was finally dried under vacuum at 65-70°C. This purification method using lead sub-acetate did not result in the same reproducibility as the barium saccharate method described below. In addition to this it became evident that the precipitation of the sucrose as barium saccharate was no more complicated than the purification of the juice with lead sub-acetate. The method finally adopted was a modification of that published by S i b l e ~For . ~ the determinations in cane juice it was found advantageous to incorporate a prepurification stage as follows. To 30 g of cane juice 1,5 g 14C sucrose were added and made up to 50 ml with distilled water. This mixture was heated to 60°C and 0,5 g barium hydroxide was added. The small amount of barium and the low temperature make precipitation of barium saccharate impossible and the precipitate found is similar to that obtained in normal lime defecation. The precipitate was centrifuged. From the clear supernatant sucrose was isolated by heating the solution to 76-80°C and adding 9,5 g of barium hydroxide. The mixture was stirred for 15 minutes. The precipitated barium saccharate was slurried in 40 ml distilled water and carbon dioxide was passed through the slurry at 70°C until neutral to phenolphthalein (external indicator). The mixture was filtered hot and the clear filtrate was made to 50 ml after which the barium saccharate precipitation was repeated. The filtrate of the second carbonatation was acidified to p H 5,8-6,O with dilute sulphuric acid. This step was incorporated to remove small quantities of residual barium salts. It was found that these barium salts, probably in colloidal form, were not removed in the subsequent ion exchange treatment. The precipitated barium sulphate was filtered through a millipore filter (0,l pm) and the filtrate was

46

Proceedings of The South African Sugar Technologists' Association-June 1973

passed through a column packed with 25 ml mixed bed ion exchange resin (Amberlite MB1). The deionised sucrose solution was concentrated to a syrup under vacuum in a rotary evaporator and the sucrose was crystallised by the addition of ethanol and by cooling. The recovered product was twice recrystallised from ethanol and dried under vacuum at 6570°C. 4.2.2 isolation and pur$cation of sucrose from molasses When the same method as described above was applied to the sucrose determination in molasses it was found that pure sucrose could not be isolated. Analysis of the final product showed that starch passed the various purification stages and therefore the procedure for sucrose in molasses was modified as follows. Five grams of molasses, 1,5 g of 14C sucrose and 10-12 ml distilled water were thoroughly mixed while being gently heated. The mixture was cooled to ambient temperature and 90 ml of absolute ethanol were added. The mixture was stirred and allowed to stand for one hour. It was then filtered on a Buchner filter through Whatman No. 5 filter paper. The clear filtrate was concentrated in a rotary evaporator to remove the ethanol and the concentrate made up to 50 ml with distilled water. No precipitation with barium hydroxide was applied to the molasses solution. The further purification was carried out by three consecutive precipitations as barium saccharate in the same way as described for juice samples. The subsequent recrystallisation from ethanol was also identical to that described for cane juice samples. 4.3 COUNTING 4.3.1 Instrument The counting instrument used in the present investigation was a Beckman p-Mate I1 scintillation counter. 4.3.2 Scintillation liquid In the earlier stages of the investigation a scintillation mixture described by Brayl1 was used. As sucrose is not soluble in nonpolar organic liquids, which are used for scintillation measurement, dioxanebased scintillation cocktails have to be utilized. Dioxane is miscible with water and the following mixture allows sufficient water to dissolve 0,2 g of sucrose, resulting in a transparent mixture. 1,4-di-2-(5-phenyl-oxazoly1)-benzene(POPOP) 0,2 g* 2,5 diphenyl oxazole (PPO) 4,o g* methanol (absolute) 100 mlt ethylene glycol 20 ml tt naphthalene 60 g** dioxane to I000 ml ** For counting 200 mg of the sucrose were dissolved in 1 ml of water and mixed with 4 ml of the scintillation liquid. This resulted in a completely transparent mixture. *BDH Scintillator tBDH Karl Fischer

ttBDH AR **Riedel-de Haan

It was found that often high counts were recorded at the initial measurement, gradually decreasing with time. This was caused by the mixture showing a high phosphorescence12 and if samples were exposed to daylight it required keeping for more than 12 hours ' in the dark before stable counts were obtained. Apart from phosphorescence another interference is caused bv chemiluminescence. an emission of light by chemich reaction. This latter mechanism is ;so enhanced by light. The effect of chemiluminescence can be overcome by heating the sample scintillation mixture in the vial for 45 minutes at 40°C before handling. This treatment, however, is not always practical. Peroxides in particular are known to cause chemiluminescence and although peroxide-free dioxane is commercially available or can be prepared, peroxides will form again when left standing in the presence of atmospheric oxygen. For more than one reason therefore toluene is to be preferred to dioxane. Toluene shows a higher counting efficiency and it does not torm peroxides, its only disadvantage being its immiscibility with water. More recently this difficulty has been overcome by the use of suitable surface active agents which can disperse a certain amount of water in toluene in such a way that a transparent mixture is obtained. A new group of solubilers has been marketed as Bio-Solvs.* According to Pollay and Stevens13 these Bio-Solvs are superior to any other presently available agents. The scintillation mixture finally adopted was made up as follows: 0,8 g PPO POPOP 0 4g Bio-Solv BBS3 15,OOml Toluene to 100,OO mlt To this mixture 10 ml of water was added. This quantity of water was experimentally found adequate to dissolve the 0,2 000 g sucrose and'result in a clear solution. For the counting procedure 0,2 000 g of sucrose was weighed accura1:ely into a counting vial and subsequently dissolved in 10 ml of the above scintillation mixture. A blank count was measured on 10 ml of the scintillation mixture and, this background count was subtracted from the sample count.

+

4.3.2 Counting vials Two types of counting vials are available for counting samples: glass and polyethylene. Due to potassium salts in the glass the background of glass vials is slightly higher than that of polythene vials. For low level counting the latter are preferable. The statement of Lew1° that plastic vials were unsuitable could not be confirmed. The polythene vials were found t o give a lower count than glass and were uniform. Within the expected accuracy the counts per minute of the standard were not dependent on the vial. *Beckman Corp

tBDH Analar

Proceedings of The South Ajiican Sugar Technologists' Association-

Although the vials are disposable it was found that it required little labour to decontaminate them for re-use. The counting rate of a blank was checked after the decontamination of a batch of vials. For decontamination the vials were boiled eight times in a solution of "Contrad"". After the final boiling the vials were rinsed with distilled water. 4.4 COUNTING ACCURACY The amount of radio-active material in the pure sucrose recovered from the sample results in a count of about 200 000 counts per minute per 0,2 g. The standard sample shows about 400000 counts per minute. Counting a sample and a standard the results shown in Table I were obtained. TABLE I

Standard counts/5 min

Sample counts/5 min

TABLE I1 Sucrose by ID and pol in juice samples

1

Sample

I

POI

F.E.J. F.E.J.

%Sucrose by I D 18,97 19,44 19,48 19,16 19,20 16,97 16,98 16,18 16,26 8,66 8.70 8;20 8,23 17,50 17,54 20,30 17,95

18,43 19,35

F.E.J.

18,94

6

M.J.

16,79

M.J.

16,11

Extract D.A.C.

8,59 8,13

Extract D.A.C. M.J.

17,28

F.E.J. M.J.

20,22 17,47

*Analyses were carried out by Huletts R & D TABLE I11

% Sucrose in molasses as determined by chemical analysis, Isotope dilution and gas chromatography

% sucrose mean 2 217 982

The standard deviation is 3 064 in the standard and in the sample 582. The errors occurring in the background count can be neglected as this is a considerably lower value and in addition frequently determined in between sample counts. The t o t a l standard deviation in the example above will be ototal = do2standard + 02sample = 3 118 This error on a count per 5 minutes of 1000 000 was considered to be sufficiently small for the purpose (0,3 % relative). By making the standard sample more radio active this error could have been reduced, but every determination would have been more expensive. 5. Results 5.1 JUICESAMPLES A number of first expressed and mixed juice samples were analysed by the I D method and by pol determination. The results are shown in Table IT. 5.2 MOLASSES Sucrose in final molasses was also determined by I D and the results were compared with those obtained by the Mackay chemical method.14 In addition the sucrose content was determined in a number of samples by quantitative gas chromatography." The results including two molasses samples supplied are listed in connection with investigations for ICUMSA in Table 111. 6. Discussion 6.1 ACCURACY OF THE METHOD In 4.4 it has been shown that the errors in counting result in a relative error of 0,3% in the final value. *BDH

Molasses sample

chemical

Isotope dilution

sz TS

PG GH

ICUMSA I (cane) SZ EM UK ICUMSA I1 (beet)

36,28 32,83 33,06 31,46 31,09 52,4 (Dutton) 53,47

1

32.94 32'83 36118 32,58 32,04 29,12 28,31 51,30 57,07

1

30y3 31,s

?Values are the mean of 5 determinations

Observation of the scatter in the results of Tables I1 and 111 indicate that other errors e.g. in weighing, pipetting, etc., increase this error. If we consider the figures in Table I11 for GH we find a mean of 37,OO with a standard deviation of 0,21 for the four determinations. 0

If we apply

-x

't we find the error for the 95 %

v n

confidence limit to be & 0,29 or 0,8 % relative. 6.2 JUICESAMPLES From Table I1 it can be calculated that for juice samples the mean difference between the values

48

Proceedings of The South Afvican Sugar Technoiqpists' Association-June

determined by I D and those by pol measurement is 0,25 % absolute. Calculation of the t value reveals that this difference cannot be caused by random errors in both determinations but is very significant. Mahoney and Lucas15 recently published results on the gas chromatographic determination of sucrose in syrups and liquors. The mean difference between sucrose by GLC in syrup and by single pol was found to be 0,50%, while this difference for double pol increased to 2,1%. This difference was checked in a number of cases by ID and it was confirmed that polarisation methods underestimate sucrose in syrups. The present results show the same trend for juice samples. Most of the analyses in Table I1 were carried out using the dioxane-containing scintillation liquid. The scatter in the results would have been less if the cocktail described in 4.3.1 had been used. SAMPLE 6.2 MOLASSES Contrary to the sucrose content in juices it was found that in molasses the conventional methods overestimate the true sucrose content. The results in Table I1 show that with one exception the sucrose content by I D is lower than that found by chemical determination. The average difference between the two methods was (GH sample excluded) 0,71, which amounts to a relative percentage of 2,15. The average difference between the chemical method and the GLC determination was 2,24% or a relative percentage of 6,8 %. The only abnormal result in this series was a sample of molasses from GH, which gave a higher

1973

sucrose value by the ID method. For this reason four independent ID determinations were carried out, but all gave a higher value than that obtained by the Mackay method. The gas chromatographic determination, however, was lower, although the difference compared to the Mackay method (0,6%) was less than the average difference.

7. Acknowledgements Thanks are due to Mr. K. J. Schaffler of Huletts R & D for carrying out sucrose analyses by GLC. REFERENCES 1. Bates, F. J., N.B.S. Bulletin (USA) C440 (1942) 127. 2. McGinnis, R. A., Beet Sugar Technology, Reinhold N. York (1951) 578. 3. Meade, G. P., Cane Sugar Handbook 9 ed., Wiley, N. York (1963) 402. 4. Hirschmiiller, H. and Hoerning, H., Z. fur die Zuckerind. (1959) 9 389. 5. Hirschmiiller, H. and Hoerning, H., Z. fur die Zuckerind. (1959) 9 499. 6. Gelen, H., Z. fiir die Zuckerind. (1970) 20 304. 7. McGagin T. A. and Eis F. G. J., Amer. Soc. of Sugar Beet Technol. (1968) 15 228. 8. Sibley, M. J., E'is, F.'G. and McGinnis, R. A., Anal. Chem. (1965) 37 1701. 9. Mauch, W., Z. fur die Zuckerind. (1970j 20 76. 10. Lew, R. Private communication, Armstar Corporation, Spreckels Sugar Division (1 971). 11. Bray, G. A., Anal. Biochem (1960) 1 279. 12. Bransome, E. D., The current status of liquid scintillation counting, Game and Stratton (1970) 88. 13. Bransome, E. D. ibidem 21 1. 14. 15th ICUMSA ~roceedinas(1970) 76. 15. Mahoney, V. C. and ~ u i a s ;P. c., Int. Sugar J. (1971) 37 291.

Smile Life

When life gives you a hundred reasons to cry, show life that you have a thousand reasons to smile

Get in touch

© Copyright 2015 - 2024 PDFFOX.COM - All rights reserved.